The long-term objective of this proposal is to understand the cellular processes that regulate high-fidelity eukaryotic DNA replication. Low fidelity replication and error-prone post-replication DNA repair can result in chromosomal aneuploidy and mutations. Knowledge of these processes is important in understanding the etiology of cancer in that cancer is the result of an accumulation of many mutations. This proposal will employ molecular genetic analysis to identify the regulatory molecules of these processes using the single-celled eukaryote, Saccharomyces cerevisiae as a model system. The combined genetic and molecular approach will focus on a number of important cell cycle protein kinases, which include Cdc7-Dbf4, Cdk1+ (cyclin-dependent kinase) and the Rad53 checkpoint kinase also known as Chk2 or Cds1.
Two specific aims are proposed.
In aim #1, the structure and function of the MCM-helicase will be analyzed by combining yeast molecular genetics with 3D-atomic X-ray crystallographic and biochemical studies of the simpler MCM-homologue from Archaea. The initial focus will be on three important cellular MCM functions: DNA binding by the central channel, oligomerization by the Zn-motifs and bypass of Cdc7-Dbf4 by mcm5-bob1. The latter function will help to understand the normal role in DNA replication played by Cdc7-Dbf4 kinase.
In aim #2, the role of these protein kinases at origins of replication in vivo will be determined by using high-resolution genomic footprinting and molecular chromatin-immunopreciptation (ChIP) techniques to probe the origin in cells with specific combination of mutations. Exploitation of the mcm5-bob1 mutant, in which replication becomes independent of Cdc7-Dbf4 kinase, will facilitate this analysis. A genome search for genes that suppress or enhance mcm5-bob1 is proposed as a way to expand the repertoire of genes important for this process. A genome-wide analysis of all 332 yeast replication origins by micro-array analysis in the mcm5-bob1 mutant will be done as a powerful way of comparing the topography of chromosome replication under different conditions.
Showing the most recent 10 out of 31 publications
